LC/IRMS analysis: A powerful technique to trace carbon flow in microphytobenthic communities in intertidal sediments

Abstract

Microphytobenthic communities are important for primary production in intertidal marine sediments. Extracellular polymeric substances (EPS), comprising polysaccharides and proteins, play a key role in the structure and functioning of microphytobenthic biofilms and allow interactions between the benthic microalgae and the associated heterotrophic bacteria. The use of stable isotopes has provided major insights into the functioning of these microbial ecosystems. Until recently, gas chromatography–isotope ratio mass spectrometry (GC/IRMS) was the principal method for compound specific stable isotope analysis in these studies. Liquid chromatography linked to IRMS (LC/IRMS) is a more recently developed technique that broadens the range of compounds that can be targeted, in particular enabling the analysis of 13C in non-volatile, aqueous soluble organic compounds, such as carbohydrates and amino acids. In this paper we present an overview of the possibilities and limitations of the LC/IRMS technique to study metabolic processes in microphytobenthic biofilms consisting of mainly diatoms. With a preliminary in-situ labeling experiment, we show that the biosynthesis of carbohydrates and amino acids in EPS and total carbohydrate and amino acid pools can be determined by LC/IRMS. Water extractable EPS were composed predominantly of carbohydrates, whereas amino acids played a minor role, both in terms of content and production. By using LC/IRMS, we will be able to quantify the biosynthesis of metabolites and, hence, to unravel in detail the metabolic pathways of the transfer of carbon from the diatoms via EPS to the bacteria.